Diabetic retinopathy is a serious complication of diabetes and is the leading cause of blindness in people with diabetes. At present, there are many views on the pathogenesis of diabetic retinopathy, including the changes of retinal microenvironment caused by high glucose, the formation of advanced glycation end products, oxidative stress injury, inflammatory reaction and angiogenesis factor. These mechanisms produce a common pathway that leads to retinal degeneration and microvascular injury in the retina. In recent years, cell regeneration therapy plays an increasingly important role in the process of repairing diseases. Different types of stem cells have neurological and vascular protection for the retina, but the focus of the target is different. It has been reported that stem cells can regulate the retinal microenvironment and protect the retinal nerve cells by paracrine production, and can also reduce immune damage through potential immunoregulation, and can also differentiate into damaged cells by regenerative function. Combined with the above characteristics, stem cells show the potential for the repair of diabetic retinopathy, this stem cell-based regenerative therapy for clinical application provides a pre-based evident. However, in the process of stem cell transplantation, homogeneity of stem cells, cell delivery, effective homing and transplantation to damaged tissue is still a problem of cell therapy.
Objective To summarize and review the heterogeneity of bone marrow derived stem cells (BMDSCs) and its formation mechanism and significance, and to analyze the possible roles and mechanisms in intestinal epithel ial reconstruction. Methods The related l iterature about BMDSCs heterogeneity and its role in intestinal epithel ial repair was reviewed and analyzed. Results The heterogeneity of BMDSCs provided better explanations for its multi-potency. The probable mechanisms of BMDSCs to repair intestinal epithel ium included direct implantation into intestinal epithel ium, fusion between BMDSCs and intestinal stem cells, and promotion of injury microcirculation reconstruction. Conclusion BMDSCs have a bright future in gastrointestinal injury caused by inflammatory bowl disease and regeneration.
OBJECTIVE To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.
Objective To investigate the heterotopic odontogenesis ability ofDelta1 gene transfected human dental pulp stem cell (DPSC) and nanohydroxyapatite/collagen (nHAC) composite scaffold. Methods The cultured human DPSC was transfected with Delta1-enhanced green fluorescent protein recombinant retrovirus supernatant，and was selected by puromycin to obtain the positive cell clone. The experimental group contained the Delta1 transfected DPSC; however, the control group did not contain the Delta1 transfected DPSC but contained DPSC transfected with vectors only. The cells were seeded into the nHAC carriers and were cultured in the odonto-inductive medium. The growth of the transduced cells in the carriers was observed by the fluorescent phase contrast microscope and the scanning electron microscope (SEM). The cell-carrier composites were subcutaneously transplanted into the Delta1 transfected 8 nude mice (female, 8 weeks old). Eight weeks after operation,the composites were taken out and tested with the histological and the immunohistological methods.Results Green fluorescence was observed inthe cells in the experimental group, which were grown in the carriers by the fluorescent phase contrast microscope. Observed by SEM, great amounts of transduced DPSC were observed along the scaffold materials, even filling the porous structures of nHAC and secreting a lot of extracellular matrix. However, in the control group, much fewer cells were found in the carriers. All the 4 Delta1 transduced DPSC-nHAC composites produced dentin-like structures that lined the surfaces of some nHAC porous structures. The odontoblast-like cells extended the cytoplasmic processes into the dentinal matrix, which was interfaced with a pulp-like interstitial tissue infiltrated with the blood vessels. Dentin sialophosphoprotein was expressed in the odontoblast-like cells when immunohisochemistry was performed. The morphology of the control composite was a typical one of the fibrous connective tissue,and only a little dentin-like structure was found in 2 of the 8 control transplants. Conclution DPSC can be used as the recipient cell of the Delta1 gene for expression and secretion of the Delta1 protein. The composites of the transfected cells and nHAC can induce heterotopic odontogenesis, which indicates that Delta1 is a novel candidate for the gene enhanced dentinpulp composite engineering.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
Objective To explore the method that can inducethe mesenchymal stem cells (MSCs) to differentiate into the neuronlike cells in vitro．Methods The neuron-like cells were isolated froman SD rat (age, 3 months; weight, 200 g). They underwent a primary culture; theinduced liquid supernatant was collected, and was identified by the cell immunohistochemistry. The C3H1OT1/2 cells were cultured, as an MSCs model, and they were induced into differentiation by β-mercaptoethanol (Group A) and by the liquid supernatant of the neuron-like primary cells (Group B), respectively. The cells were cultured without any induction were used as a control (Group C). Immunohistochemistrywas used to identify the type of the cells． Results The result of the immunochemistry showed that the cells undergoing the primary culture expressed the neurofilament protein (NF) and the neuronspecific enolase (NSE), and they were neuron-like cells. β-mercaptoethanol could induce the C3H1OT1/2 cells toexpress NF and NSE at 2 h, and the expression intensity increased at 5 h. The liquid supernatant of the primarily-cultured neuron-like cells could induce theC3H1OT1/2 cells to express NF and NSE at 1 d, but the expression intensity induced by the liquid supernatant was weaker than that induced by β-mercaptoethanol. The positivity rate and the intensity expression of NSE were higher than those of NF. Conclusion MSCs can differentiate into the neuron-like cells by β-mercaptoethanol and the microenvironment humoral factor, which can pave the way for a further study of the differentiation of MSCs and the effectof the differentiation on the brain trauma repair.
Objective To explore the relationship of the limited resource of the autologous bone marrow mesenchymal stem cells (MSCs) in articularcavity to the treatment results of full-thickness articular cartilage defect, and to investigate whether the extrogenous sodium hyaluronate(SH) promotes the migration of MSCs cultured in vitro tothe articular defect in vivo. Methods Sixty-six Japan rabbits were made the model of the full-thickness articular cartilage defect (5 mm width and 4 mm depth).The autologous MSCs were extracted from the rabbit femur, cultured in vitro, labeledby Brdu, and injected into the injured articular cavity with or without SH. Theexperiment was divided into 4 groups; group A (MSCs and SH, n=15); group B (MSCs, n=15); group C (SH, n=18); and group D (non-treatment, n=18). The morphologic observation was made by HE staining, Mallory staining and immunohistochemical staining after 5 weeks, 8 weeks and 12 weeks of operation. Results There were significant differences in the thickness of repairing tissue between group A and group B(Plt;0.01); but there were no significant differences between group A and group C, and between group B and group D(P＞0.05). Thehistological observation showed that the main repairing tissue was fibrocartilage in group A and fiber tissue in group B. Conclusion MSCs cultured in vitro and injected into the articular cavity can not improve the treatment results of the articular cartilage defect. Extrogenous SH has effect on repairing cartilage defect. The extrogenous SH has no effect on the chemotaxis of the MSCs, and on the collection of MSCs into the joint defect.
ObjectiveTo determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration. MethodsHair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups. ResultsThe isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences (P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant (P<0.05). ConclusionLentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.
Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.